prolong gold anti fade reagent with dapi  (Cell Signaling Technology Inc)

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide facilitates macrophage-Treg crosstalk to mitigate chronic inflammation in diabetic wound healing

doi: 10.1016/j.bioactmat.2025.09.044

Figure Lengend Snippet: GelMa/SCS-MN promotes re-epithelialization and revascularization in diabetic wounds. (a) Photographs of wounds of diabetic mice at different time after treatments. (b) Quantitative analysis of the relative residual wound areas at different times (n = 6). (c) Sagittal sections of day 3 and day 7 wound tissue immunolabeled for cytokeratin (red), integrin a5 (Itga5, green) and DAPI (blue). Scale bar, 200 μm. (d) Sagittal sections of day 7 wound tissue immunolabeled for cytokeratin (red), Itga5 (green) and labeled with 5-ethynyl-2′-deoxyuridine (EdU, gray) (proliferation), cell nucleus for DAPI (blue). Scale bar, 100 μm. (e) Quantitative statistics of the migration distance of epidermal tongue from wound edge (n = 5). (f) Quantitative statistics of the number of EdU + epidermal cells in each visual field (n = 5). (g) Sagittal sections of day7 wounds tissue immunolabeled for Endomucin (Emcn, red) and EdU (green), cell nucleus for DAPI (blue). Scale bar, 200 μm. (h) Quantitative statistics of the Emcn + vascular gap in wound (n = 5). (i) Representative H&E staining of diabetic wound tissue on day7 and day 14. The green line indicates epidermal gap. Scale bar, 200 μm. (j–k) Quantification of (j) epidermis gap on day 7 and (k) scar distance on day 7 and day 14 according to HE staining images (n = 3). (l) Sagittal sections of day14 wounds tissue immunolabeled for Emcn (red), cell nucleus for DAPI (blue). Scale bar, 200 μm. Data are presented as mean ± SD and statistical significance was analyzed via one-way ANOVA with Tukey's multiple comparison test for (f), (h) and (j), two-way ANOVA with Sidak's multiple comparison test for (b), (e), and (k). P value: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Article Snippet: The specimens were sectioned to a thickness of 10 μm, washed three times with TBST, and mounted using ProLong gold anti-fade reagent with DAPI (Cell Signaling Technology).

Techniques: Immunolabeling, Labeling, Migration, Staining, Comparison

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide facilitates macrophage-Treg crosstalk to mitigate chronic inflammation in diabetic wound healing

doi: 10.1016/j.bioactmat.2025.09.044

Figure Lengend Snippet: Lacking Tregs impairs SCS-mediated diabetic wound healing. (a) Schematic illustrating experiment depleting Tregs. (b) Flow cytometric quantification of Tregs (Foxp3 + ) in the diabetic skin wound after anti-CD25 mAb injection (n = 6). (c) Representative wound images of diabetic mice after the indicated treatments and the corresponding time-course quantification of relative residual wound area (n = 8). (d) Sagittal sections of day 7 wound tissue immunolabeled for cytokeratin (red), integrin a5 (Itga5) (green), EdU (gray) and DAPI (blue). Scale bar, 200 μm. (e) Quantitative statistics of the migration distance of epidermal tongue from wound edge (n = 8). (f) Quantitative statistics of the number of EdU + epidermal cells in each visual field (n = 12). (g) Representative flow cytometric plots of CD31 + Emcn + cells expression in diabetic wound tissue on day7 post-wounding and corresponding quantitative results (n = 6). (h) Representative immunostainings of CD31 (red), Emcn (green), EdU (gray) and DAPI (blue) in diabetic wound tissue on day7 post-wounding. Scale bar, 200 μm. (i) Representative H&E staining of diabetic wound tissue on day7 and day14. Scale bar, 500 μm. (j–k) Quantification of (j) epidermis gap on day 7 and (k) scar distance on day14 according to H&E staining images (n = 3). Data are presented as mean ± SD and statistical significance was analyzed via two-way ANOVA with Sidak's multiple comparison test for (c), unpaired two-tailed Student's t -test for (b), (e), (f), (g), (j) and (k). P value: ∗P < 0.05, ∗∗P < 0.01.

Article Snippet: The specimens were sectioned to a thickness of 10 μm, washed three times with TBST, and mounted using ProLong gold anti-fade reagent with DAPI (Cell Signaling Technology).

Techniques: Injection, Immunolabeling, Migration, Expressing, Staining, Comparison, Two Tailed Test

Journal: Infection and Immunity

Article Title: Rickettsia heilongjiangensis suppresses RIPK1 kinase-mediated host cell death during the infection

doi: 10.1128/iai.00158-25

Figure Lengend Snippet: Rh -B8 infection-induced host cell apoptosis in vivo and in vitro . (a) Survival analysis of Ifnar -/- mice infected with Rh -B8 ( n = 9) compared to control mice injected with SPG buffer ( n = 6). All mice died within one week following intravenous injection of Rh -B8. (b) Spleen tissue from Rh -B8-infected Ifnar -/- mice exhibited enlargement and more severe pathological changes compared to uninfected mice. TUNEL staining on splenic paraffin sections revealed a multitude of apoptotic cells (red). Scale bar, 20 µm. Nuclei were indicated by blue DAPI staining. (c) Bacterial counts in tissues, including the spleen, liver, kidney, lung, and brain from five Rh -B8-infected Ifnar -/- mice, were quantified using plaque assay. Means are shown with error bars representing ±SEM. (d) During infection at a multiplicity of infection (MOI) of 1.0, Rh -B8 exhibited slow propagation in the early stages of infection, followed by exponential growth starting at 48 hours post-infection (hpi). In parallel, the viability of HMEC-1 cells decreased significantly by 72 hpi. Data are expressed as mean ± standard deviation (SD), with each experiment conducted in biological triplicates, and results were averaged across all replicates. (e) Immunofluorescence microscopy images demonstrated an increase in the number of apoptotic HMEC-1 cells during Rh -B8 infection. Apoptotic cells were labeled using TUNEL staining (green), nuclei were visualized with DAPI (blue), and bacteria were detected using an anti-OmpB antibody (red). Scale bars, 20 µm.

Article Snippet: Finally, the cell side of the coverslip was inverted onto a microscope slide containing a DAPI anti-fade reagent (Beyotime, P0131).

Techniques: Infection, In Vivo, In Vitro, Control, Injection, TUNEL Assay, Staining, Plaque Assay, Standard Deviation, Immunofluorescence, Microscopy, Labeling, Bacteria

Journal: Infection and Immunity

Article Title: Rickettsia heilongjiangensis suppresses RIPK1 kinase-mediated host cell death during the infection

doi: 10.1128/iai.00158-25

Figure Lengend Snippet: Inhibition of caspase-8 does not sensitize Rh -B8-infected HMEC-1 cells to necroptosis. (a and b) Rh -B8-infected cells treated with the caspase-3 inhibitor (Z-DEVD-FMK) showed a significant reduction in cell death during the late stages of infection. An immunofluorescence assay showcased images of TUNEL (green) and DAPI (blue) in HMEC-1 cells infected with Rickettsia (red) at 72 hpi. The growth curve illustrates the replication of Rh -B8 and cell viability of HMEC-1 treated or untreated with Z-DEVD-FMK. (c and d) Rh -B8-infected HMEC-1 cells treated with the caspase-8 inhibitor (Z-IETD-FMK) did not show protection against apoptosis at 72 hpi. The replication of Rh -B8 and cell viability of HMEC-1 between the Z-IETD-FMK-treated and untreated groups exhibited no significant difference. (e and f) The activity of RIPK1 kinase, blocked by necrostatin-1, also did not influence rickettsial growth or host cell viability. (g) The Western blot assay demonstrated that HMEC-1 cells infected with Rh -B8 and treated with the RIPK1 inhibitor necrostatin-1 (Nec-1) at various stages continued to undergo apoptosis. Representative results from three independent experiments are presented for the blotting assays. Uninfected cells treated with each inhibitor for 72 hours were included as controls in panels (a), (c), and (e). Data in (b), (d), and (f) were shown as mean ± SD (each time point has three biological replicates). *** P < 0.001 relative to normal infection, P -values were calculated using an unpaired t -test (two-tailed).

Article Snippet: Finally, the cell side of the coverslip was inverted onto a microscope slide containing a DAPI anti-fade reagent (Beyotime, P0131).

Techniques: Inhibition, Infection, Immunofluorescence, TUNEL Assay, Activity Assay, Western Blot, Two Tailed Test